Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies

• DOI: 10.1261/rna.1887910
• Publication Date: 2010-05-25T04:12:11Z
• Authors: Ying Poi Liu; Monique A. Vink; Jan-Tinus Westerink; Eva Ramirez de Ar...; Pavlina Konstanti...; Olivier Ter Brake; Ben Berkhout
• Keywords: Ribonuclease III; MicroRNAs; 0303 health sciences; 03 medical and health sciences; RNA, Untranslated; Genetic Vectors; Lentivirus; RNA Interference; Promoter Regions, Genetic; 3. Good health

RNAi-based gene therapy is a powerful approach to treat viral infections because of its high efficiency and sequence specificity. The HIV-1-based lentiviral vector system suitable for the delivery RNAi inducers HIV-1 susceptible cells due ability transduce nondividing cells, including hematopoietic stem stable transgene into host cell genome. However, presence anti-HIV short hairpin RNA (shRNA) microRNA (miRNA) cassettes can negatively affect titers. We show that shRNAs, which target genomic RNA, strongly reduced titers but inhibition pathway via saturation could rescue production. miRNAs in genome (sense orientation) results minor titer reduction Drosha processing. A major cause miRNA vectors incompatibility cytomegalovirus promoter with system. Replacement this an inducible resulted almost complete restoration titer. also showed antisense poly(A) signal sequences have dramatic effect on These not all are compatible care should be taken design encoding inducers.